Process of preparing liver extracts



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a aliases vsuccess or rnsraanvo Howard L. Ede-ii, Ghicasailll...asslanor to our o and Company, chicane, lit, a corporation of Illinois No Drawina'.

This invention relates to processes of prepa g liver extracts, and itincludes processes wherein ground livers are subjected to extractionwith warm water, the extract concentrated to pasty form} the paste takenup in an aqueous alcohol solution, and the alcoholic solutionrefrigerated at a temperature of at least about minus 26 F.

or below, i and the alcoholic solution of antianemlc principle decantedor filtered from impurities separated out at the low temperature stated.e

Liver concentrates for use as therapeutic agents.

are customarily prepared by subjecting ground animal livers toextraction with water heated to about 18o" F. after the extractionprocess is completed the mixture of aqueous extract and residue isfiltered, or the liquid extract separated from the residue in equivalentways. The extract is then concentrated to a paste by evaporation in.vacuo, aqueous alcohol solution of ahout '70 per cent strength isadded, the solution filtered. and the filtrate concentrated to a finalpaste in which the anti-anemic value in one cram of the paste isequivalent to about ninety-five grams of orieninTal fresh liver startingmaterial. This liver paste can. then be administered orally in cansulesortalren up in inocuous solvents usually alcoholic to'aet a liquidconcentrate for oral administration.

illi these liver extracts, and concentrated pastes ob tained therefrom,can, however, be removed when the paste is made-alcoholic and'thealcohol solution subjected to extremely low temperature, at least as lowas minus 15 F. and generally as low as minus 20 F. I have discoveredthat at these low temperatures the proteins, glycogen, dextrins andotherimpurities crystallize out, and that the cold alcoholic solutioncan bereadily decanted from the impurities.

or the mixture or precipitated impurities and I have now discovered thatthe impurities in Application February 15, rest, Eel No. earn d Claims.(Cl. lollidi v alcoholic solution can be filtered. This way oiseparating residual impurities from ordinary concentrated liver pasteshas many advantages, chiei anions which is the fact that. nothing hasbeen added to the paste which can lessen the activity of the therapeuticprinciples in the paste. Thus I avoid the use oi chemical reagents whichmay seriously modify, destroy, or adulterate the antianemic principlesextracted from the livers.

Moreover, my final paste or concentrate has a much higher concentrationwith respectrto the. original starting liver. By that i mean, the finalpaste of my invention is such that 1 gram thereof represents about 235grams of the fresh liver. Consequently, I can prepare solutions forhypoderuiatic injection containing about 25 percent of totalsolids'wherein 1 cc. of the solutions represents the anti-anemicsubstances derived from illu grains of liver.

In practicing my invention '1 first prepare a liver paste in thewell-known way by extracting around animal livers with hot water havinga temperature oi about ldd i The extract, after separation of insolubleresidue, is then concentrated to a paste in vacuo, as usually done inthe art, and the paste talren up in aqueous alcohol the liver iscoasulated and does not appear in the extract. However, the extract doescontain sisnificant amounts of glycogen and dextrin, and smaller amountsof proteins which impurities, as stated, are removed by my invention.

To each lilo grams of the paste prepared and described alcove I thensidda'bout all!) cubic centimeters oil a lil percent solution ofalcohol; This alcoholic solution can he made up from puregrain alcohol,but ordinarily it is more economical to use a denatured alcohol. Onesuch denatured alcohol of commerce consists of so percent ethyl alcohol,in percent methyl alcohol and 5 percent water. The denatured alcohol is,or course, diluted with water until its alcoholic content amounts toabout to percent by volume. For best results the strength or the alcoholsolution should be about 70 percent,-but this can vary from about 60 to80 percent by volume. After adding the -no further precipitation occurs.

aqueous alcohol to the paste the mixtureis then well stirred and placedin a cooler operating at a temperature of not more than about minus 20F. The mixture is kept in the cooler for about 16 to 20 hours duringwhich time the remaining proteins, glycogen, dextrin, small quantitiesof lipoid materials, and other impurities precipitate out. The alcoholicmixture is kept refrigerated until Aqueous alcohol of the concentrationstated completely dissolves the anti-anemic principle of liver even atthe low temperature stated, but undesirable impurities precipitate orcrystallize out of solution. The supernatant alcoholic layer can bereadily decanted or filtered from the precipitate, such separation, ofcourse, being performed at the low temperature used for chilling.During. the rei'rigeration, 'I can allow the temperature to increase bytwo or three degrees, but I do not obtain the benefits of my inventionunless the temperature during the chilling is minus 15 F. or lower.

. The decanted alcoholic solution is thenevaporated under vacuo to athick syrup containing about 66 percent of total solids. At this stagethe product is biuret negative, indicating that no proteins are present,but the product still contains small quantities of reducing substances'which may possibly be glucose. The syrup is next warmed until itsconsistency is morefluid.

To each 100 grams or. the warm syrup thus formed I add about 800 cubiccentimeters ofabsolute ethyl alcohol. The syrup is best added to thealcohol while stirring vigorously, and stirring is continued until theinsoluble solids in the mixture become light in color. The mixture isthen allowed to settle, the alcoholdecanted from the solids or filteredon, and the solidsthen dried on pans in vacuo at the usual low absolutepressures, 28 inches or less, employed in this art.

The resulting pasty material amounts to about one-third of the originalstarting paste. Its con-- water at a temperature or about 180 1".,concentrate thewaterextracttoapaste,takeupthe paste in 70 percentaqueous alcohol solution and then directly proceed to chill thissolution at minus 20 1!. The subsequent steps of decanting or filteringthe alcohol solution while at minus 20 concentration to a syrup,admixture oi the syrup with absolute alcohol and recovery of the finalproduct are identical with those given in the preceding example.

It is understood that variations in the proportlons of alcoholicsolutions used can be resorted to.v Obviously those skilled in the artwill understand that the amount of aqueous alcohol used shouldbesufllcient to form fluid mixtures which, after chilling, can be decantedor filtered. Consequently, I do not wish to be limited to any particularproportions.

Having thus described my invention, what I claim is:

1. In the process of preparing liver extracts, the steps of. freeingsuch extracts if impurities which comprise admixing a paste of the watersoluble constituents of the liver with an aqueous alcohol solutionhaving an alcoholic strength or about 60 to 80 .percent by volume,cooling the mixture to a temperature of not more than minus I cent byvolume.

4. The process as in claim 1 wherein the cooling temperature is minus 20F. and the alcohol solution has a strength of about 70 percent byvolume.

5. The process 01'. obtaining a liver extract which comprises admixing apaste of the water soluble constituents of liver with an aqueous alcoholsolution having a strength of about 60 to '80 percent by volume,chilling the mixture to a temperature or not more than minus 15 F.maintaining the chilled solution at-said temperature to insureprecipitationof substances insoluble in the solution at saidtemperature, separating the alcoholic solution from the solids,evaporating the alcoholic solution in vacuo to a syrup, admixing thesyrup with absolute alcohol, removing insoluble material from thealcohol solution, and drying the insoluble material in vacuo. 1

6. The process as in claim 5 wherein the cooling temperature is minus 20F.

7. The process as in claim 5' wherein the alcohol solution has astrength of about 70 per--v cent by volume.

8. The process as in claim 5 wherein the cooling temperature is minus 20F. and the alcohol solution has a strength or about 70 percent byvolume. a

HAVARD L. KEIL.

